Intestinal stem cells (ISCs) replenish the intestinal epithelium every few days and represent a general model for tissue differentiation and stem cell properties. Differentiation of ISCs and their immediate progeny into mature cells requires precise control of genes that are expressed in each stage. Although mature intestinal cells could always be obtained in large numbers to study gene regulation, ISCs were, until recently, difficult to isolate. Discovery of the ISC marker Lgr5 has made it possible to isolate and characterize this cell population, which is also important to regenerative medicine and the study of intestinal cancers. Homeostasis of the intestinal epithelium is sustained by rapidly dividing Lgr5+ ISCs. One crucial factor for gene activation in differentiated intestinal cells is the transcription factr Cdx2 (caudal type homeobox 2), however its role in ISCs was previously unknown. My analysis of Cdx2-/- intestines reveals that ISCs lacking Cdx2 are significantly impaired in replication and in the long term unable to give rise to mature cell types. Thus, Cdx2 seems to serve distinct roles in ISCs and their terminally differentiated progeny and this difference is reflected in my detection of Cdx2 binding at distinct enhancer and promoter elements in Lgr5+ ISCs and mature intestinal villus cells in vivo. This proposal builds on these preliminary data to address the specific role of Cdx2 in ISCs. In Aim 1 I will test the hypothesis that Cdx2 directly regulates key signaling pathways required for ISC proliferation. To reveal Cdx2's direct transcriptional targets in ISCs in vivo, I will integrate data from Cdx2 chromatin immunoprecipitation (ChIP-seq) in ISCs with global mRNA transcript profiling (RNA-seq) of wild-type and Cdx2-/- mouse ISCs. As my preliminary data point to epidermal growth factor (EGF) signaling as a strong direct Cdx2 target pathway, I will use mouse intestinal organoid cultures to additionally test the specific hypothesis that Cdx2 controls ISC proliferation through EGF signaling. In Aim 2 I will determine whether Cdx2 loss, which limits ISC proliferation, also limits the formation of tumors (adenomas) that originate specifically in ISCs. Although Lgr5+ ISCs are the likely cell of origin for intestinl tumors, including human colorectal cancer, the role of Cdx2 in the disease is confusing. To test the hypothesis that tumor formation is significantly compromised in the absence of Cdx2 function, I will conditionally remove both Cdx2 and Apc (a negative regulator of the tumor-promoting Wnt signaling pathway) in Lgr5+ ISCs. This strategy will address the controversy about Cdx2 functions in intestinal tumors in a genetic model system. Together, Aims 1 and 2 have the potential for significant impact on understanding of ISC regulation, which can be applied to promising stem cell-based regenerative therapies as well as improved understanding of colorectal cancer, the second leading cause of cancer mortality in the United States.